The invention relates to methods for synthesizing compounds for the selective modification of nucleic acids in biological compositions.
The transmission of viral diseases (e.g., hepatitis A, B, and C, acquired immunodeficiency syndrome, and cytomegalovirus infections) by blood or blood products is a significant problem in medicine. Other biological compositions, such as mammalian and hybridoma cell lines, products of cell lines, milk, colostrum, and sperm, can also contain infectious viruses. Screening donor biological compositions for viral markers can help reduce the transmission of viruses to recipients, but many screening methods are directed to only a few discrete viruses, and are therefore incomplete, and may also be less than 100% sensitive. It is therefore important to inactivate viruses contained in donor blood, blood products, or other biological compositions.
A number of agents that are capable of inactivating viruses in blood have been developed. For example, ethyleneimine monomer and ethyleneimine oligomers (including dimers, trimers, and tetramers) are very effective viral inactivating agents. Methods for using ethyleneimine oligomers for inactivating viruses in biological compositions are described in U.S. Ser. No. 09/005,606 (filed Jan. 12, 1998) now U.S. Pat. No. 6,093,564, hereby incorporated by reference.
In general, the invention provides a method for synthesizing ethyleneimine dimer (1-aziridineethanamine) which includes reacting 2-(2-aminoethylamino)ethanol with an aqueous HX solution, where X is a halogen, to produce N-(2-haloethyl)-1,2-ethanediamine dihydrohalide, reacting the N-(2-haloethyl)-1,2-ethanediamine dihydrohalide with a base in a solvent to convert the N-(2-haloethyl)-1,2-ethanediamine dihydrohalide into ethyleneimine dimer, and then purifying the dimer from the solvent (e.g., by continuous extraction).
In preferred embodiments, the halogen is bromine (most preferred), chlorine, fluorine, or iodine. In another preferred embodiment, the HX is diluted, such that the solution is 30-55% (w/w) HX. In yet another preferred embodiment, the solvent includes a C1-6 alcohol (e.g., ethanol or methanol) or water.
In other preferred embodiments, the HX is added drop-wise to the 2-(2-aminoethylamino)ethanol, the temperature of the 2-(2-aminoethylamino)ethanol is less than 10xc2x0 C. during the addition of the HX, and the reaction of 2-(2-aminoethyl amino)ethanol with HX includes the step of refluxing. Preferably, the yield of the N-(2-haloethyl)-1,2-ethanediamine dihydrohalide from this reaction is at least 50% of the theoretical yield. More preferably, the yield is at least 75% of the theoretical yield.
In still other preferred embodiments, reacting the N-(2-haloethyl)-1,2-ethanediamine dihydrohalide to produce ethyleneimine dimer includes the steps of refluxing and distillation. Preferably, the yield of ethyleneimine dimer from this reaction is at least 20% of the theoretical yield. More preferably, the yield is at least 25% of the theoretical yield, and most preferably, the yield is at least 30% of the theoretical yield.
Preferably, the ethyleneimine dimer which results from the method of synthesis is at least 90% pure, more preferably at least 95% pure, and most preferably at least 98% pure.
The method of synthesis described herein provides several advantages over previous methods: (1) The starting compounds are all relatively inexpensive; (2) the yield of product is greater than 20% of the theoretical yield; and (3) the steps of synthesis are easy, inexpensive and amenable to large-scale production. All of these advantages allow for less expensive production of ethyleneimine dimer.
Other features and advantages of the invention will be apparent from the following detailed description and from the claims.
An example of the synthesis is provided below. From the description provided herein, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.